The production and purification of recombinant polypeptides such as pharmaceutical proteins in a highly purified and well-characterized form, has become a major task within the area of protein engineering in general, and in the pharmaceutical industry in particular.
The preparation of such recombinant polypeptides relies frequently on techniques which involve the production of the polypeptides as fusion proteins or hybrid proteins, wherein a protein or polypeptide of interest is fused to a carrier or a fusion partner such as a polypeptide or a protein.
The presence of a fusion partner or carrier which is fused to the polypeptide of interest has the advantages that it may render the fusion protein more resistant to proteolytic degradation, may facilitate enhanced expression and secretion, improve solubility and allow for subsequent affinity purification of the fusion protein. Also by fusion protein expression, potentially bio-hazardous materials, such as peptide hormones, may be produced in an inactive form which can then be activated subsequently in vitro by cleaving off the fusion partner.
However, such fusion proteins themselves are not normally suitable as end products as the fusion partner e.g. may affect the biological activity or stability of the polypeptide of interest and, if the protein is to be used clinically, may cause antigenicity problems. Therefore it is necessary to cleave the fusion protein to release the polypeptide of interest.
In principle this can be achieved by chemical or biochemical methods such as enzymatic cleavage. However, it is important that the cleavage is highly specific and only takes place in a cleavage sequence between the polypeptide of interest and the fusion partner, i.e. the junction region, but preferably not within the polypeptide of interest itself, as this may e.g. severely affect the bioactivity of the polypeptide of interest. Such methods employ agents that act by hydrolysis of peptide bonds and the specificity of the cleavage agent is determined by the identity of the amino acid residue at or near the peptide bond which is cleaved.
Biochemical methods for cleavage of fusion proteins are based on the use of proteases (proteolytic enzymes). However, enzymatic cleavage of fusion proteins is limited in that the amino acid(s) which are specific for the cleavage site can at the same time also occur in the polypeptide of interest itself. Therefore, enzymes are particularly suitable which, in order to cleave, not only recognises one amino acid but rather a sequence of amino acids, since the probability that a particular amino acid sequence is present once again in the polypeptide of interest in addition to the cleavage site between the polypeptide of interest and the fusion partner is less the larger the number of amino acids necessary for the recognition and cleavage of the cleavage sequence.
Up till now, a number of proteases have been used for enzymatic cleavage of fusion proteins by contacting the fusion protein with a protease under appropriate conditions.
WO 03/010204 relates to a process for separating a polypeptide of interest from a fusion protein by the use of ubiquitin cleavage enzyme, which according to this document is an enzyme that cleaves a peptide bond next to the amino acid sequence RGG at the C-terminus of proteins such as ubiquitin.
U.S. Pat. No. 6,010,883 disclose a method wherein blood clotting factor Xa (EC 3.4.21.6; a S1 serine-type peptidase formed from the proenzyme factor X by limited proteolysis) is used for cleaving off a fusion partner from a fusion protein. This protease specifically cleaves after the amino acid sequence X-Y-Gly-Arg, wherein X is Ile, Leu, Pro or Ala, and Y is Glu, Asp, Gln or Asn. Factor Xa preferably cuts after the cleavage sequence Ile-Glu-Gly-Arg.
Other prior art enzymes which have been suggested and used in methods for specific cleavage of fusion proteins include tobacco etch virus Nla proteinase, collagenase, enterokinase, subtilisin and thrombin.
However, several problems may be encountered when using proteolytic cleavage in fusion protein systems. One major problem is the occurrence of non-specific proteolytic attack of the fusion protein which results in cleavage at several locations and consequently product loss and generation of contaminating fragments. Also problems with inefficient or incomplete cleavage of the fusion protein frequently occur with the presently known enzymes. Such inefficient cleavage reduces the yield and may also introduce heterogeneity to the purified protein resulting in the recovery of only a small fraction of the desired protein.
A further problem that is associated with several of the presently applied enzymes for fusion protein cleavage is that spurious or extraneous amino acids are frequently attached to the cleaved polypeptide product (the polypeptide of interest). These amino acids are typically present when a linker is cleaved, and the unrelated amino acid residues may have an effect on the properties of the resulting polypeptide of interest. This may be critical for proteins produced for human therapeutics. Therefore, it is highly desirable to be able to produce pure authentic polypeptides free of extraneous amino acid short sequences or residues.
The problem with extraneous amino acids remaining on the polypeptide of interest after cleavage is illustrated in U.S. Pat. No. 4,543,329 which describes a process for selectively cleaving a fusion protein by the use of collagenase. However, the use of this enzyme produces a polypeptide of interest with the extraneous amino acid sequence Gly-Pro at its N-terminal. In order to obtain the polypeptide of interest in authentic form, these extraneous amino acids (Gly and Pro) must subsequently be removed in a further reaction step by the use of one or more different amino peptidases (such as aminoacylproline amino peptidase and proline amino peptidase).
The problem is also illustrated in U.S. Pat. No. 5,427,927 which describes a process for sequence specific cleavage of fusion proteins by the use of IgA protease, wherein a IgA protease recognition site is inserted in the junction region of a fusion protein which is subsequently cleaved with IgA protease. The recognition site for the IgA protease is the amino acid sequence Y-Pro↓X-Pro (SEQ ID NO: 58), in which X can be any amino acid, Y can be one or several arbitrary amino acids, and ↓ denotes the cleavage site. However, the proteins of interest which are formed after cleavage by IgA protease, are characterised by having an X-Pro extraneous amino sequence at its N-terminal, i.e. the resulting polypeptide of interest is not in its native or authentic form.
Presently, the most widely used proteolytic enzymes for fusion protein cleavage are the serine proteases factor Xa and thrombin. However, both enzymes are known to perform non-specific cleavage of fusion proteins. In addition, factor Xa has to be isolated from bovine serum and as a consequence when it is used to cleave proteins for therapeutic applications an extensive purification and analysis is necessary afterwards in order to detect pathogenic factors such as viruses and prions which may be present (e.g. prions causing bovine spongiform encephalopathy). Furthermore, these enzymes are rather expensive.
In view of these prior art shortcomings and drawbacks, it is therefore an object of the present invention to provide an improved method for enzymatic cleavage of fusion proteins.
It has been found by the present inventors, that the above technical problems may be overcome by using Granzyme B protease (EC 3.4.21.79) for enzymatic cleavage of fusion proteins. Thus, it has surprisingly been found that Granzyme B protease allows for highly efficient cleavage of fusion proteins having a Granzyme B protease cleavage site with a high degree of cleavage specificity. In particular Granzyme B protease has proven to perform more specific fusion protein cleavage than the presently and widely used protease factor Xa. It has furthermore been found that Granzyme B cleavage of fusion proteins that contain a Granzyme B recognition sequence positioned between an N-terminal fusion partner and a C-terminal polypeptide of interest, wherein the cleavage site is adjacent to the polypeptide of interest, results in a polypeptide of interest that have no extraneous amino acids derived from the cleavage site, i.e. a polypeptide in authentic form. Thus, recombinant proteins of interest with native amino acid sequence may be produced as a result of fusion protein cleavage by Granzyme B. Also, the Granzyme B protease has the advantage that it can be produced recombinantly. Finally, it has been found that by substituting the Cysteine amino acid no. 228 (chymotrypsinogen numbering) with Phenylalanine in human Granzyme B, a higher final protein recovery can be obtained when producing recombinant human Granzyme B.